Immersion objectives have been known for a long time from practical use. The idea underlying such objectives is that image quality in terms of light intensity and resolution can be utilized by optimally exploiting the so-called angular aperture of an objective. In order to achieve optimum utilization of the information available in the light that is to be analyzed, it is most common to use immersion liquids, which improve the optical transition between a preparation and the objective. The immersion liquid increases the total refractive index of the objective lens and thus its numerical aperture, which can be calculated from the refractive index of the immersion liquid and the sine of half the angular aperture.
Immersion liquids are liquids whose refractive index is close to the refractive index of the glass used for the sample slide and lenses. If an air gap between an entrance or exit lens of a microscope objective (hereinafter called simply an “objective lens”) and a sample slide is filled with an immersion liquid of this kind, the numerical aperture of the objective is increased. This results in increased light intensity, and furthermore permits increased resolution for the objective.
Liquids are preferably used as immersion media, for example water, oil, or glycerol. Liquids having substances added to them are also a possibility, for example caffeine in order to decrease bacterial development in the water. Surfactants are also used. It is also possible to use immersion gases. When immersion gases are used, they are allowed to flow through continuously, at a certain temperature and flow rate, between the objective lens and the objective specimen slide or specimen.
Depending on the refractive index of the immersion medium, a more or less perfect optical adaptation to the refractive indices of the objective lenses and the objective carrier, which are usually manufactured from glass, can be achieved. With such a “perfect” adaptation, the otherwise usual light losses triggered by refraction and/or total reflection at the specimen slide are almost entirely eliminated, but at least very considerably reduced. In practice, the objectives to be used, called “immersion objectives,” are optimized for the use of specific immersion liquids. A distinction is thus made between oil objectives, for which oil is used as an immersion medium; water objectives, for which water is used as an immersion medium, for example for the investigation of water-containing preparations such as living cells; and glycerol objectives, for which the immersion medium is a glycerol that is similar to the embedding medium of a corresponding preparation.
Immersion liquids are usually combined into groups, specifically in terms of different mixtures which are notable for the fact that by mixing different liquids, the optical refraction index of the immersion medium can be varied and thus adjusted.
When an immersion liquid is used, it is essential that it be located between the objective or outer lens and the preparation or specimen slide glass, in order to achieve mutual optical adaptation of the two components Immersion liquid is usually applied onto the objective lens and/or onto the specimen slide shortly before the actual experiment.
The immersion liquid is usually applied manually, with the aid of a pipette, onto the objective lens or the sample slide. This is difficult with high-resolution microscopes, however, since with these the objective focus is located very close to the objective lens, for example at a distance of 0.2 mm therefrom. The spacing between the objective lens and the sample slide will therefore be only fractions of a millimeter in the case of high-resolution microscopes, and it is not readily possible to introduce immersion liquid from outside, using a pipette, into such a narrow gap. Instead, the objective must be moved away from the sample slide before application of the immersion liquid, and then moved back toward the sample slide. This movement can be realized, for example, by pivoting the objective or moving it up and down. This makes application of the immersion liquid particularly laborious.
A further difficulty arises with automated microscopes, in which the objective is scanned automatically over the sample slide so that a large number of samples can be investigated automatically in a short time. When the sample slide is moved relative to the objective, it may happen that the immersion film detaches, and operation of the microscope is thereby disrupted. Automated microscopes are also used to acquire images of samples, for example living cells, over a long period of time. In this case the immersion medium will partially evaporate and must therefore be replenished from time to time; this is likewise laborious when done manually.
It would be particularly advantageous, however, to use an immersion liquid specifically in the context of automated high-performance microscopes, with which the use of an immersion medium is cumbersome, because of the improved image quality thereby achievable, in particular the elevated light intensity and greater resolution capability.
Regarding documents of the existing art, reference may be made purely by way of example to DE 202 05 080 U1 that describes, separately, an immersion objective having a shielding element. What is provided therein, more precisely, is a sealing ring in the form of a bellows that is arranged in the region between the objective and the specimen. It serves to receive a small quantity of immersion liquid, and retains it in the region relevant for the beam path. The shielding element holds the immersion liquid at the point where it is required, and reduces to a minimum both evaporation and the “consumption” of immersion liquid.
Reference is also made to WO 02/093232 A2. This document likewise describes the use of an immersion objective; in this case a delivery apparatus is provided for automatic delivery of immersion medium into the region between the outer surface of the sample slide and the exit lens of the objective. Concretely, the delivery device comprises a delivery tube connected to the objective, which tube conveys the immersion medium precisely into the relevant region. The tracking system necessary for this occupies a very considerable amount of space, and requires that the delivery tube extend into the region between the exit lens and the sample or sample slide. Thus not only is the known apparatus complex in terms of design, but its utilization is problematic because of the considerable physical size, specially when multiple objectives are to be made available for selection, especially in the context of automatic operation.
The known apparatus encompasses, concretely, a delivery tube that is mounted laterally on the objective and through which an immersion liquid is delivered into the vicinity of the center of the objective lens. From the exit opening of the delivery tube, the immersion liquid is then pulled by capillary forces into the gap between the objective lens and the sample slide.
To ensure that enough immersion liquid is always present, in the known apparatus the liquid is continuously delivered in excess, and excess immersion liquid is collected in a drainage channel that is provided on an objective head of the objective, and aspirated with an aspiration device.
For automatic microscopy in particular, it is advantageous if resolutions can be adapted by rapidly changing the objectives that are used. The objective change can be made manually or automatically in that context. An automatic changeover between immersion objectives, simultaneously with continued automatic delivery of immersion liquid, is not possible with any of the systems mentioned above.
In addition, it is often necessary in practical use to switch between different immersion media or immersion liquids, for example oil and water. During the switchover from one immersion liquid to another, it is therefore necessary for the particular immersion liquid being used to be removed as thoroughly as possible from the objective and the specimen slide. This has hitherto been possible only manually. The immersion liquid is usually applied manually or by way of a cumbersome gestural system. This requires considerable time, and the process of applying or introducing the immersion liquid is imprecise. It also always entails a risk of contamination, especially since some immersion media are toxic. Manual handling of the immersion media is to be avoided for this reason as well.
As stated earlier, the systems known from practical use are of only limited suitability for practical utilization in microscopy. Care must always be taken to ensure that such systems have no quality-reducing disadvantages. While the provision of a replenishment fitting in the gap between the objective and the specimen slide is inconvenient, a replenishment system attached to the objective consistently causes a torque acting on the objective. In cases where the immersion objectives are arranged in an objective turret, rotation of the objective turret is impeded by replenishment systems of this kind. All of the aforesaid disadvantages are not tolerable, especially in the context of automatic operation.
Be it noted further that when immersion liquid is used, preference is given to certain types of objective in which the lens on the specimen-slide end, i.e. the exit lens of the objective, is located closest to the specimen slide. Given the surface tension there, capillary forces are utilized in order to introduce the immersion medium into the region between the outer lens and the specimen. The utilization of capillary forces is advantageous even though they depend on numerous boundary conditions, for example on the surface tension of the components and constituents involves, in particular the surface tension of the immersion liquid and the interfacial tension of the immersion liquid with the surface of the specimen slide and the surface of the exit lens. If the surfaces that are to be thoroughly wetted with the immersion liquid are highly contaminated, for example as a result of fingerprints on the specimen slides or encrusted immersion-oil residues on the specimen slides and/or on the exit lens, it may be assumed that the necessary capillary forces will not take effect. The risk correspondingly exists that the gap between the exit lens and the specimen slide will be only incompletely wetted with immersion oil. If the relevant active optical surfaces are only partly wetted with immersion oil, this has a quality-reducing effect on the microscope images obtained.
A further quality-reducing feature is air bubbles that can occur in the gap between the specimen slide and the objective. Such air bubbles decrease the capillary forces and prevent complete wetting of the optical surfaces with immersion liquid. The creation of small air bubbles is promoted by the use of filling tubes to supply the gap with immersion liquid. The risk exists in particular at higher flow rates, which have a kind of atomization effect due to the entrainment of air.
An immersion objective is described in U.S. Pat. No. 3,202,049, in which a particular device is provided for delivering immersion liquid into the region between the specimen or a specimen slide and the outer lens (exit lens) of the objective. This device encompasses a cap, surrounding the objective body and open in the region of the outer lens, that is screwed with an internal thread onto an external thread of the objective body. The cap forms a gap toward the outer lens, a small reservoir for receiving immersion liquid being formed inside the cap. The immersion liquid can emerge from the reservoir through the gap.
Provision of the cap has the advantage that as compared with the otherwise known systems, it is physically extremely small, i.e. arranged in rotationally symmetrical fashion around the objective body. It contains a repository of immersion liquid that is kept on hand, for operation of the immersion objective, in accordance with the volume provided therein.
The known system utilizes gravity and manual rotation of the cap in order to bring about emergence of the immersion liquid from the annular gap formed between cap and objective. It is correspondingly necessary for such immersion objectives to be used exclusively in conventional fashion, specifically such that the objective “looks down” onto the sample being investigated. The known gravity-dependent immersion objective cannot be used for inverted microscopy, in which the objective “looks” from below into the specimen slide or onto the material. The known immersion objective is also not suitable for the automatic use of multiple immersion objectives, especially since continual manual adjustment of the quantity of immersion liquid to be discharged is not possible during automatic operation. Lastly, the known immersion objective has the further disadvantage that large quantities of oil build up on the preparation or slide because of constant emergences of immersion liquid. When oils are used, encrustation cannot be avoided because of constant evaporation.